By using plasmids, foreign genes have been transferred into host cells of various microorganisms including Escherichia coli (referred to as E. coli hereinafter). So as to allow the characteristic properties of a foreign gene to be exerted in host cells in a more stable manner, attempts have been made to integrate a foreign gene into a host chromosome by using a lysogenic phage.
Meanwhile, L. casei Shirota strains, YIT9018 and YIT9029, have been utilized widely in for example lactic acid beverages and fermented milk, and it is revealed that these strains have excellent physiological actions, particularly antitumor activity, blood cholesterol reducing action, hypotensive action and anti-ulcer effect (Japanese Patent Laid-open Nos. 113718/1980, 5236/1992, 25055/1993, and 116155/1994).
A number of reports have been issued about shuttle vectors in hosts such as these lactic acid bacteria, particularly L. casei (see Japanese Patent Laid-open Nos. 128692/1990, 259086/1991, and 58890/1992). Most of these shuttle vectors in reports have been prepared via transformation as plasmids, so not any of these shuttle vectors has been satisfactory in terms of stability.
According to a conventional method for transferring a foreign gene into a host cell by utilizing a lysogenic phage, not only the objective foreign gene but also DNA sequences unnecessary after the transfer are simultaneously transferred. For example, a site-specific recombination enzyme gene region, a chemically resistant gene region necessary for recombinant selection, and a replication origin functioning in E. coli are simultaneously integrated into the chromosome of a host, but functionally, these regions are not necessary after the transfer of the gene.
From the standpoint of the subsequent utilization of a transformant, these regions functionally unnecessary after the transfer of the gene may sometimes be disadvantageous.